Production of folding-defective proteins underlies the pathogenesis of many heritable disorders in man, yet the unique features of misfolded proteins and the cellular machinery that recognizes them remain largely unknown. The long-term goal of this project is to identify the cellular factors that couple the recognition of folding-defective proteins in the endoplasmic reticulum to their degradation by a process known as ER-associated degradation (ERAD). Despite considerable progress made in linking the cytoplasmic ubiquitin-proteasome system (UPS) to ERAD, fundamental questions remain unanswered. Foremost amongst these is the nature of the cis and trans signals that identify a protein as an ERAD substrate and the molecular events that divert proteins from the biosynthetic folding pathway to ERAD. Previous work from my lab and many others has firmly established a central role for the UPS in the targeting of misfolded ER proteins for degradation. The immediate goal of this project is thus to identify the cellular machinery by which malfolded proteins in the ER are recognized. We will use a combination of functional genomics and traditional cell biology to identify E3 Ub ligases and other critical elements of the UPS that collaborate in the recognition of ERAD substrates. To this end, three specific aims are proposed. In the first aim we plan to screen libraries of small hairpin interfering RNA (shRNA) directed against all probable UPS components in the human genome. Hits from this screen will be validated by a battery of assays and the interaction of the newly identified components with substrate and other ERAD machinery will be studied in detail. Preliminary data are included identifying Hrd1 as an E3 ligase for degradation of unassembled AMPA-type glutamate receptor subunits, thereby validating the efficacy of our functional genomics screen. Thus, the second aim is to elucidate the cis- and trans- acting factors that promoting Hrd1- dependent degradation of GluR1 subunits. The third aim complements the first by performing a comprehensive biochemical and microarray investigation of how cellular stress response pathways respond to topologically and structurally distinct classes of ERAD substrate. It is anticipated that this analysis will provide new understanding of how cells respond to physiologically relevant protein stress, and will facilitate the identification of new components of that direct specific classes of substrate to degradation by ERAD.